Integrated illumination-detection flow cell for liquid chromatography

ABSTRACT

A liquid chromatography flow cell including an integrated light source and an integrated detection chamber. The integrated light source includes a plurality of light emitting diodes (LEDs), wherein each LED emits light of a specific wavelength. The light emitted from the integrated light source is directed to pass through a sample in a flow chamber of the flow cell without any optical conditioning, and the light not absorbed by the sample flows out of the flow chamber directly into the integrated detection chamber, where an intensity of the unabsorbed light is measured by detectors coupled to the integrated chamber.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application claims priority to U.S. Provisional ApplicationNo. 62/462,786, entitled “INTEGRATED ILLUMINATION-DETECTION FLOW CELLFOR LIQUID CHROMATOGRAPHY,” and filed on Feb. 23, 2017, the entirecontents of which are hereby incorporated by reference in their entiretyfor all purposes.

BACKGROUND AND SUMMARY

Conventional liquid chromatography systems, such as high-performanceliquid chromatography (HPLC), two-dimensional HPLC, and ultra-highperformance liquid chromatography (UHPLC), operate on the principle ofexposing a sample to a particular wavelength of light to determinephysical properties of the sample. Light emitted from a light source isoptically transmitted through a flow cell of the liquid chromatographysystem. The light transmitted through the flow cell may be partially tofully absorbed by the sample inside the flow cell. Any unabsorbed lightmay scatter through the sample and is transmitted to a detection system(for example, to a variable wavelength detector system or to a diodearray detector system). Based on the detection profile, a constituentelemental or compound makeup of the sample is generated.

The light sources used for HPLC systems may include deuterium, mercuryarc, and/or tungsten lamps, which are relatively large compared to atypical flow cell of the HPLC system. Additionally, the light emittedfrom these light sources is spatially and spectrally broad, thusrequiring optical conditioning. Considerable heat is also generated bythe above-mentioned light sources, which may lead to undesirable thermaleffects on the sample inside the flow cell. The light sources also haveto be pre-warmed before use and typically have a limited life (forexample, a tungsten lamp is typically replaced after ˜2000 hours ofuse), increasing both the time and the cost of operating the HPLCsystem.

One example approach to address the above-mentioned problems has beenshown by Bland et al. in U.S. 2014/0191117 A1. Therein, a plurality oflight emitting diodes (LEDs) are used as a light source in achromatography system, wherein each LED emits a light of a differentwavelength. The LEDs do not require pre-warming before use and lastlonger than the light sources mentioned above.

However, the inventors herein recognize that in the above-mentionedapproach, multiple optical elements, including spectral filters,alignment and focusing lenses, a broadband filter, a beam splitter,etc., may be present between the light source and the flow cell, whichadds substantial optical pathlength, leading to a large form factor ofthe HPLC system. A detector of the HPLC system may be positioned todetect the light scattered after it flows through the sample in the flowcell, which may further increase the size of the HPLC system. Themultiple optical elements may also contribute to additional noisegeneration, thereby diminishing the signal-to-noise ratio.

The inventors herein have recognized the above-mentioned issues inliquid chromatography systems and have engineered a way to at leastpartially address them. In one example approach, a detector systemincludes a flow cell including an optically transparent first wall andan optically transparent second wall, the optically transparent secondwall positioned opposite the optically transparent first wall; aplurality of light sources integrated within the flow cell, theplurality of light sources configured to emit light to travel throughthe optically transparent first wall into the flow cell; and a detectionchamber integrated with the flow cell and configured to capture lightpassing out from the flow cell through the optically transparent secondwall into the detection chamber. In this way, a form factor of thedetector system may be reduced while a signal-to-noise ratio isincreased.

The plurality of light sources may be each controlled independently by acontroller. In one example, each of the plurality of light sources is anLED. Each of the plurality of light sources may be configured to emitlight of a specific wavelength that flows through the opticallytransparent first wall into the flow cell and from the flow cell throughthe optically transparent second wall into the detection chamber. Eachof the plurality of light sources is adjacent to a reference diodeintegrated within the flow cell, wherein a signal from the referencediode is relayed to the controller, which in turn controls the outputlevel of the light source. Furthermore, the detection chamber mayinclude one or more photodetectors coupled thereto as well as a diffuse,reflective interior coating. The interior coating may enable widerdispersion light exiting the flow cell through the optically transparentsecond wall to be fully collected and measured by the one or morephotodetectors. In this way, the signal-to-noise ratio of the detectorsystem may be further increased while the form factor of the system isfurther decreased. Furthermore, the use of LEDs, which do not have to bepre-warmed and have a long lifespan, may enhance efficiency and reducethe cost of operating the detector system, which may be included in aliquid chromatography system.

The above advantages and other advantages and features of the presentdescription will be readily apparent from the following detaileddescription when taken alone or in connection with the accompanyingdrawings.

It should be understood that the summary above is provided to introducein simplified form a selection of concepts that are further described inthe detailed description. It is not meant to identify key or essentialfeatures of the claimed subject matter, the scope of which is defineduniquely by the claims that follow the detailed description.Furthermore, the claimed subject matter is not limited toimplementations that solve any disadvantages noted above or in any partof this disclosure.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows a schematic depiction of a high-performance liquidchromatography (HPLC) system.

FIG. 2 shows a schematic depiction of a liquid chromatography flow cell.

FIG. 3 illustrates a first embodiment of an integratedillumination-detection flow cell.

FIG. 4 illustrates a second embodiment of an integratedillumination-detection flow cell.

FIG. 5 illustrates a method for operating a liquid chromatographydetector system including an integrated illumination-detection flowcell.

FIG. 6 illustrates an example of a liquid chromatography flow cell inwhich a light source and a sample detector is not integrated.

FIG. 7 shows schematic of a first configuration of optical componentsrelative to the liquid chromatography flow cell.

FIG. 8 shows schematic of a second configuration of optical componentsrelative to the liquid chromatography flow cell.

FIG. 9 shows a schematic of a third configuration of optical componentsrelative to the liquid chromatography flow cell.

FIG. 10 shows schematic of a dichroic beam combining system that may beincluded in a liquid chromatography detector system.

FIG. 11 shows schematic of a fiber-coupled beam combining system thatmay be included in a liquid chromatography detector system.

FIGS. 12A and 12B illustrate schematics of a waveguide beam combiningsystem that may be included in a liquid chromatography detector system.

FIG. 13 shows schematic of an integrating chamber beam combining systemthat may be included in a liquid chromatography detector system.

FIG. 14 shows schematic of a polygonal prism beam combining system thatmay be included in a liquid chromatography detector system.

FIG. 15 shows schematic of a compound beam combining system that may beincluded in a liquid chromatography detector system.

FIG. 16 shows schematic of a multiplexing light source selection systemthat may be included in a liquid chromatography detector system.

DETAILED DESCRIPTION

The present description is related to a detection system, which may beincluded in an HPLC system, such as the example HPLC systemschematically shown in FIG. 1. FIG. 2 shows an example configuration ofa flow cell that may be included in the detection system. The flow cellmay contain a liquid sample. The liquid sample in the flow cell mayabsorb the light at least partially, and the remaining unabsorbed lightmay be directed to flow toward a detector, such as an ultraviolet (UV)and/or visible light (VIS) photodetector. FIGS. 3 and 4 illustrate afirst embodiment and a second embodiment, respectively, of an integratedillumination-detection flow cell, in which the detectors and the lightsource are both integrated with the flow cell to decrease a number ofoptical elements between the light source and the liquid sample and theliquid sample and the detectors, which reduces the form factor of theHPLC system. A method for operating the integratedillumination-detection flow cells is illustrated in FIG. 5. FIG. 6illustrates a HPLC flow cell with a single wavelength light source,which may be arranged according to the example configurations shown inFIGS. 7-9. FIGS. 10-16 show examples of multiple wavelength illuminationsystems that may be included in a detector system of an HPLC system.

Referring to FIG. 1, a schematic diagram of an example HPLC system 100is shown. HPLC system 100 includes a control system 110, a solventreservoir 120, a pump 130, a sample injector 140, a column 150, adetection system 160, a fraction collector 170, and a waste container180. Multiple components of the HPLC system 100 may be included in acommon housing 101. As shown in FIG. 1, the pump 130, sample injector140, column 150, and detection system 160 are all housed within commonhousing 101. However, in other examples, more or fewer components couldbe housed in the common housing. For example, the solvent reservoir,fraction collector, and/or waste container may be housed in the commonhousing. Additionally or alternatively, the control system 110 may behoused in the common housing.

The control system 110 is communicatively coupled to other components ofthe HPLC system (as indicated by dashed lines), as described furtherbelow, in order to send and receive signals during system operation.Control system 110 may include a controller, such as a desktop or laptopcomputer, one or more user input devices (e.g., a mouse, keyboard, touchscreen), a display system, and/or a communication system operable tocouple the controller to one or more remote computing devices, forexample. Control system 110 may receive input from an HPLC systemoperator to initiate a sample run. In other examples, the sample run maybe automated or semi-automated, with control system 110 initiating thesample run according to one or more methods stored in a memory of thecontrol system. The controller may be an electronic controller and mayinclude a memory storing instructions executable to carry out one ormore of the methods described herein. Furthermore, the controller mayinclude one or more physical logic devices, such as one or moreprocessors, configured to execute instructions. Additionally oralternatively, controller 12 may include hardware or firmware configuredto carry out hardware or firmware instructions. The memory may includeremovable and/or built-in devices, including optical memory,semiconductor memory, and/or magnetic memory. The memory may includevolatile, nonvolatile, dynamic, static, read/write, read-only,random-access, sequential-access, location-addressable,file-addressable, and/or content-addressable devices. The memory andlogic device(s) may be integrated together into one or morehardware-logic components, such as field-programmable gate arrays(FPGAs).

Prior to sample injection, HPLC system 100 may be primed with solvent.Control system 110 may activate pump 130, which draws solvent fromsolvent reservoir 120 that is fludically connected to pump 130 and othercomponents of HPLC system 100 downstream of pump 130 by lines. Solventreservoir 120 may hold one or more solvents, such as hexanes, ethylacetate, dicholormethane, and methanol, with the solvent(s) pumped bypump 130 input into control system 110 by the HPLC system operator orautomatically selected based on a pre-programmed method stored in thememory of control system 110. In one example, one solvent, such ashexanes, may be used to prime HPLC system 100. In another example, twosolvents at a selected ratio, such as 4:1 hexanes:ethyl acetate or 9:1dichloromethane:methanol, may be used. Other suitable solvents may beselected to suit the application chemistry, chromatography method,column type, etc. In still another example, three or more solvents maybe used. The solvent(s) and ratio used may be selected (e.g., by theHPLC operator or control system 110) based on the components to bepurified. Thus, as used herein, the term “solvent” also includes solventmixtures. The term solvent refers to the mobile phase eluate exiting thecolumn without analyte.

Solvent pumped by pump 130 flows through sample injector 140 and intocolumn 150. Column 150 may contain a solid phase adsorbent, such assilica gel, alumina, or other functionalized medium, selected based onthe components to be analyzed. The length and diameter of column 150 mayalso be selected based on the application chemistry or chromatographymethod and may be installed by the HPLC system operator prior toactivating the pump. After flowing through the column, the solvent flowsthrough detection system 160. Detection system 160 may include one ormore light sources, a flow cell, and one or more photodetectors, asdescribed further herein, although other types of detection systems mayadditionally or alternatively be used, such as photoionizationdetectors, charged aerosol detectors, electrical conductivity detectors,electrochemical detectors, mass spectrometers, refractive indexdetectors, etc. In the example of FIG. 1, detection system 160 isconfigured to measure UV and visible light transmission and absorbance.Detection system 160 may measure a baseline absorbance value of thesolvent. Control system 110 may subsequently subtract this baselineabsorbance value from values measured after sample injection. Afterflowing through detection system 160, the solvent is flowed to wastecontainer 180.

Once HPLC system 100 is primed (e.g., the column is equilibrated withthe appropriate solvent), a sample 145 may be injected via sampleinjector 140 into the flow path of solvent pumped by pump 130. In someexamples, sample injector 140 may be an autosampler programmed to injecta sample according to a pre-determined method executed by control system110. In another example, the HPLC operator may manually operate sampleinjector 140.

Once sample 145 is injected, it is loaded (e.g., adsorbed) onto theresin of column 150. Different components of sample 145 may havedifferent affinities for the resin as well as the solvent flowingthrough column 150. Thus, components with higher affinities for theresin will move through the column more slowly, while components withhigher affinities for the solvent will move through the column morequickly. For example, if the resin is silica gel and the solvent has alow polarity (such as hexanes or a solvent mixture with a high ratio ofhexanes), a more polar component will have stronger interactions withthe silica gel and will be retained on the column for a longer duration,and a more nonpolar component will have stronger interactions with thesolvent and will be eluted from the column after a shorter duration.Further, the solvent(s) used may be adjusted throughout the sample run,such as by increasing the polarity of the solvent mixture, in what isknown as a gradient elution. In other examples, the composition of thesolvent may remain constant throughout the sample run in what is knownas isocratic elution. Other elution methods may also be used, suchstepwise elution or combination elution methods.

After each component of sample 145 is eluted from column 150, it passesthrough detection system 160. Detection system 160 exposes the componentto one or more wavelengths of light, as described further herein. Aslight from a light source of detection system 160 passes through thecomponent, which is diluted in the solvent, some or all of the light maybe absorbed, with the amount of light transmitted through the componentmeasured by a detector of detection system 160. Control system 110 maygenerate an absorbance profile of the component from data received fromdetection system 160. From detection system 160, each component may flowto fraction collector 170. Fraction collector 170 may fill collectioncontainers, such as vials or test tubes, with eluted components. Thecontainers may be filled to a set volume, with the fraction collectoradvancing to the next container when the set volume is reached. Inanother example, the fraction collector may advance to the nextcontainer based on the absorbance profile of the component that haspassed through the detector. If the absorbance profile changes, controlsystem 110 may trigger fraction collector 170 to advance to the nextcontainer, as a change in absorbance profile may indicate a differentcomponent. Thus, two components may be kept separate. Filled containersmay be referred to as fractions.

Control system 110 may generate a chromatogram with absorbance (asmeasured by detection system 160) as the Y-axis plotted againstretention time (the time it takes a component to pass through HPLCsystem 100) and/or fraction number as the X-axis. The chromatogram maycontain distinct peaks in absorbance corresponding to each analyte(e.g., component) that has passed through the system. Optimally, theabsorbance signal is proportional to the concentration of analyte, andthe peaks for each analyte are separated. The HPLC system operator mayidentify fractions containing a component of interest based on thechromatogram and/or absorbance profiles. Therefore, the ability toidentify fractions containing the component of interest may depend onthe accuracy and sensitivity of detection system 160.

Referring now to FIG. 2, a schematic shows a flow cell 200, which may beincluded in a detection system of a liquid chromatography system (forexample, in detector system 160 of HPLC system 100 of FIG. 1). The flowcell 200 may include a chamber 210 defined by a first section 202 and asecond section 204, opposite the first section 202 of the flow cell 200.In addition, a first optically transparent window 206 and a secondoptically transparent window 208, opposite the first opticallytransparent window 206, may define the chamber 210. In other examples,when flow cell 200 is a sample cell, the chamber 210 may be a cuvettewhere the first optically transparent window 206 may be a firstoptically transparent wall of the cuvette and the second opticallytransparent window 208 may be a second optically transparent wall of thecuvette.

A liquid sample (e.g., sample 145 of FIG. 1) may enter the chamber 210along an entry path 216. In one example, the entry path 216 may passthrough the second section 204 of the flow cell and may open into thechamber 210, delivering a liquid sample into the chamber. The liquidsample may flow into the chamber 210 and flow out of the chamber throughan exit path 218. In one example, the exit path 218 may pass through thefirst section 202 of the flow cell 200, as shown. In another example,the exit path 218 may pass through the second section 204 of the flowcell 200. A first gasket 212 along the first optically transparentwindow 206 and a second gasket 214 along the second opticallytransparent window 208 may prevent the liquid sample from leaking fromthe chamber 210.

Light may enter the chamber 210 through the first optically transparentwindow 206 and travel through the chamber 210, containing the liquidsample, as indicated by a dashed arrow 220. The light entering thechamber originates from a light source (not shown). In one example, thelight source may be a plurality of LEDs, each LED of the plurality ofLEDs emitting a light of a different wavelength (for example, 254 nm,280 nm, 395 nm, 525 nm, etc.). Light sources and their configurationwithin the detector unit will be described below with reference to FIGS.3-4 and 7-16.

As light travels through the chamber 210 and the liquid sample therein,the liquid sample (e.g., comprising one or more analytes and one or moresolvents) may at least partially absorb the light. Light absorption bythe liquid sample depends on constituents present in the sample. Thelight that is not absorbed by the liquid sample exits the chamber 210through the second optically transparent window 208. The unabsorbedlight exiting the second optically transparent window may then bedirected to a sample detector (not shown). The sample detector may be avariable wavelength detector or a diode array detector, for example.

Thus, FIG. 2 shows a flow cell that is separate from a light source anda sample detector. Furthermore, multiple optical elements, such aslenses, mirrors, filters, slits, and beam splitters, may be presentbetween the light source and the flow cell and/or the flow cell and thesample detector. As a result, the optical pathlength may be increased,increasing a form factor of a corresponding detector system and HPLCsystem. Furthermore, a signal-to-noise ratio of a light signal receivedby the sample detector may be decreased. Therefore, systems thatminimize the optical pathlength and/or increase an amount of lightcaptured by the sample detector may decrease a form factor of thecorresponding detector system and HPLC system as well as increase thesignal-to-noise ratio at the sample detector.

FIG. 3 illustrates a first embodiment of an integratedillumination-detection flow cell 300. As an example, the integratedillumination-detection flow cell 300 may be included in a detectionsystem of an HPLC system, such as detection system 160 of HPLC system100 shown in FIG. 1. The integrated illumination-detection flow cell 300may include a flow cell 308 with a flow channel 306, similar to the flowcell 200 illustrated in FIG. 2. The flow channel 306 may include aninlet 322 and an outlet 324, wherein a liquid sample may flow in throughthe inlet 322 along a path indicated by a dashed arrow 325 through theflow channel 306 and exit through the outlet 324. A flow rate of theliquid sample through the flow channel 306 may be regulated by a flowregulator (not shown) upstream of the inlet 322. The flow channel 306may be at least partially inside the flow cell 308.

The flow cell 308 may include a plurality of diode pairs 311, each ofthe diode pairs 311 including a light source 312 and a photodiode 310adjacent to the light source 312. The plurality of diode pairs 311 areintegrated with the flow cell 308, such as positioned within an interiorof a housing of the flow cell 308. In one example, the plurality ofdiode pairs 311 may be coupled to a bottom surface 315 of the flow cell308, wherein the bottom surface 315 of the flow cell is parallel to theflow channel 306, as illustrated in FIG. 3. Each light source 312 may bea light-emitting diode (LED), wherein each LED in the plurality of diodepairs 311 emits light of a different wavelength (for example, 254 nm,280 nm, 395 nm, 525 nm, etc.). The LEDs do not have to be pre-warmed andare longer lasting than other light sources, such as a tungsten lamp, amercury arc lamp, and/or a deuterium lamp. Each LED may be selected andcontrolled independently to emit light of desired wavelength, forexample, by a controller 350 coupled to the integratedillumination-detection flow cell 300. The controller 350 may be includedin control system 114 of HPLC system 100 shown in FIG. 1, for example.The LEDs may receive power from a power source coupled to the LEDs (notshown). In another example, each light source 312 may be a laser diode,with each laser diode 312 emitting light of a different wavelength. Instill other examples, the light source 312 may comprise a LED in one ormore of the plurality of diode pairs 311, while the light source 312 maycomprise a laser diode in a remaining number of the plurality of diodepairs 311.

The photodiode 310 adjacent to the light source 312 of each diode pair311 may be a photodetector, for example. Each photodiode 310 may serveas a reference detector for the corresponding light source 312 of thediode pair 311, and each of the plurality of diode pairs 311 may becoupled to the controller 350. The controller 350 may control an outputlevel/intensity of light emitted by the light source 312 of the diodepair 311 based on an intensity of a signal received from the photodiode310 paired with the activated light source 312, as will be discussed indetail with reference to FIG. 5. For example, as an amount of lightmeasured by the photodiode 310 increases, a voltage output by thephotodiode 310 may increase.

Light emitted from each light source 312 may travel directly toward theflow channel 306 along a plurality of light paths 316. The light emittedfrom each light source 312 of each diode pair 311 may be incident on afirst optically transparent wall 319 along the flow channel 306. A firstportion of light from the light paths 316 may pass through the firstoptically transparent wall 319 and into the flow channel 306, while asecond portion of light from the light paths 316 may be reflected off ofthe first optically transparent wall 319 and may travel along aplurality of light paths 314 to the photodiode 310 of the diode pair311, as illustrated in FIG. 3. The amount of light detected by thephotodiode 310 is proportional to the amount of light being coupled intothe flow channel 306. Although the light paths 316 and 314 are onlyillustrated with respect to one of the diode pairs 311, it should beunderstood that similar light paths are present for each of theplurality of diode pairs 311.

The integration of the light sources 312 along the bottom surface 315 ofthe flow cell 308 enables light emitted by each of the light sources tobe incident directly on the first optically transparent wall 319. In oneexample, the bottom surface 315 of the flow cell including the lightsource 312 may be parallel to the first optically transparent wall 319.A distance d1 separating the bottom surface 315 and the first opticallytransparent wall 319 may enable the light emitted by the light source312 integrated with the bottom surface 315 to travel directly to thefirst optically transparent wall without any optical conditioning. Alength L1 of the first optically transparent wall may entirely overlapwith a length L3 of the bottom surface, such that the light emitted fromeach of the light sources may be incident on the first opticallytransparent wall, and not on optically opaque wall sections 321 and 323of the flow channel, which are adjacent to the first opticallytransparent wall 319 of the flow channel. The integration of the lightsources within the flow cell may be such that the light path 316 and thelight path 314 may not be obstructed by any structure present inside theflow cell. In one example, as shown in FIG. 3, no coupling optics may bepresent between the light source 312 and the first optically transparentwall 319.

Upon entering the flow channel 306 through the first opticallytransparent wall 319, the light may be at least partially absorbed bythe liquid sample inside the flow channel 306. The absorption of lightmay depend on constituents present in the liquid sample in the flowchannel, as further described above with respect to FIGS. 1 and 2. Theunabsorbed light may exit the flow channel through a second opticallytransparent wall 318, opposite the first optically transparent wall 319.The second optically transparent wall 318 may be parallel to the firstoptically transparent wall 319 without being in face sharing contactwith the first optically transparent wall 319. In one example, a lengthL2 of the second optically transparent wall 318 may be equal to thelength L1 of the first optically transparent wall 319, and the secondoptically transparent wall 318 may be aligned with the first opticallytransparent wall 319 such that the second optically transparent wall 318extends in parallel along an entire length of the first opticallytransparent wall 319.

Optically transparent glass, optically transparent plastic, fusedquartz, etc., may form the first optically transparent wall and thesecond optically transparent wall. In one example, the transparency ofthe first optically transparent wall 319 and the second opticallytransparent wall 318 may exceed eighty percent. In an example, when theflow channel 306 is comprised of a capillary, the optical transparencyof a first wall of the capillary receiving the light emitted by the LEDand the optical transparency of a second wall, opposite the first wall,may be based on optical characteristics of the material used to make thefirst and the second wall of the capillary (for example, opticallytransparent glass, plastic, quartz, etc.). In another example, the flowchannel 306 may instead be a cuvette, where in the liquid sample isloaded into the cuvette though an opening instead of flowing through theflow cell 308. In such an example, the first optically transparent wall319 may be a first wall of the cuvette, and the second opticallytransparent wall 318 may be a second wall of the cuvette, opposite andparallel to the first wall.

Light may exit the second optically transparent wall 318 along aplurality of light paths 320 into a detection chamber 302, which may beconfigured to collect the light from the plurality of light paths 320.The detection chamber 302 is directly coupled to and integrated with theflow cell 308, such as by sharing the second optically transparent wall318. For example, a bottom wall 330 of the detection chamber 302 may beat least partly the same as the second optically transparent wall 318(e.g., the bottom wall 330 may be at least partly formed by the secondoptically transparent wall 318), which may at least partially define anupper surface of the flow cell 308. The light passing out from the flowchannel 306 through the second optically transparent wall 318 maydirectly enter the detection chamber 302 along the plurality of lightpaths 320. The detection chamber 302 is coupled to the flow cell suchthat the light passing through the second optically transparent wall 318leads directly to the detection chamber 302, without anystructures/optical elements along the light paths 320 between the secondoptically transparent wall 318 and the detection chamber 302. Thedetection chamber 302 may be optically opaque and leak-proof, except atthe second optically transparent wall 318, to enable all of the lightalong the light paths 320 to be captured in the detection chamber 302.

The detection chamber 302 includes one or more photodetectors coupledthereto. As shown, detection chamber 302 is coupled to a calibratedultraviolet (UV) detector 305 and a calibrated visible light (VIS)detector 304. The UV detector 305 may be configured to measure light ofa fixed wavelength or a range of wavelengths within the UV portion ofthe electromagnetic spectrum (e.g., between 190 nm and 300 nm).Similarly, the VIS detector 304 may be configured to measure light of afixed wavelength or a range of wavelengths within the visible lightportion of the electromagnetic spectrum (e.g., between 300 nm and 600nm). Thus, both of the UV detector 305 and the VIS detector 304 serve assample detectors, measuring sight transmitted through the liquid samplewithin the flow channel 306. The light entering the detection chamber302 (e.g., via the second optically transparent wall 318) may vary inits degree of dispersion depending on a density of the liquid sample inthe flow channel 306, a relative amount of scattering and absorptionthrough the liquid sample, and an effective refractive index of theliquid sample in the flow channel 306. Therefore, detection chamber 302may include a small (e.g., 1-inch cubical or cylindrical) integratingchamber 303 to allow wider dispersion light exiting the flow cell 308(e.g., via the flow chamber 306 and the second optically transparentwall 318) to be fully collected and measured by the UV detector 305and/or the VIS detector 304. The integrating chamber 303 may include adiffuse, reflective coating along the interior of detection chamber 302that creates a uniform scattering or diffusing effect, preserving anoptical power of the reflected light. Thus, detection chamber 302 mayalso be referred to as an integrating detection chamber.

The light detected by each of the UV detector 305 and the VIS detector304 in the detection chamber 302 is the light transmitted through theliquid sample inside the flow chamber 306 (e.g., not absorbed by theliquid sample). Absorbance (A) may be related to the transmittance (T)by the equation A=−log₁₀(T), which may be used by the controller 350 toconvert a transmittance signal from one or both of the UV detector 305and the VIS detector 304 into a sample absorbance measurement at adesired wavelength. The absorbance measurement may then be plotted as afunction of time to generate a chromatogram representing the absorptionprofile of the liquid sample inside the flow cell. Furthermore, theabsorbance measurement may be related to sample concentration by theBeer-Lambert law, A=εcl, where A is the absorbance; E is a molarextinction coefficient (e.g., molar absorptivity), which is an intrinsicproperty of a chemical species; c is the concentration; and l is apathlength of the sample. In the example of FIG. 3, the pathlength isthe distance between the first optically transparent wall 319 and thesecond optically transparent wall 318.

FIG. 4 shows a second embodiment of an integrated illumination-detectionflow cell 400, which may be used in a liquid chromatography system(e.g., HPLC system 100 of FIG. 1). The integrated illumination-detectionflow cell 400 may share common features and/or configurations as thosealready described above with reference to FIG. 3. Components previouslyintroduced in FIG. 3 are numbered similarly and not reintroduced.

The configuration of the flow channel 306 along the flow cell 308 in theintegrated illumination-detection flow cell 400 is the same as describedabove with reference to FIG. 3, except for an optical element betweeneach light source 312 and the first optically transparent wall 319 ofthe flow channel 306. The flow cell 308 includes the plurality of diodepairs 311 along the bottom surface 315, wherein each of the diode pairsincludes the light source 312 and the reference diode 310. Light emittedfrom each light source 312, which may be an LED emitting light ofspecific wavelength, may travel toward the flow channel along a lightpath 416 to one of a plurality of coupling optics 430. Each of thecoupling optics 430 may be positioned between each light source 312 andthe first optically transparent wall 319 of the flow channel 306. Eachof the coupling optics 430 directs (e.g., collimates or focuses) thelight emitted by the corresponding light source 312 to the firstoptically transparent wall 319, as indicated by light paths 417,increasing an amount of light entering the detection chamber 302 via theflow channel 306. Some of the light emitted by each light source 312 maybe reflected off the corresponding coupling optic 430 and may travelalong light paths 414 to the photodiode 310 of the diode pair 311, asillustrated in FIG. 4.

Each of the coupling optics 430 may be a prism, a free-form optic, aFresnel lens, a window, or a ball lens. The coupling optics 430 mayminimize dispersion/scattering of the light emitted by the light sourcewhile travelling from the corresponding light source 312 to the firstoptically transparent wall 319. Due to the inclusion of the couplingoptics 430, a distance d2 between the bottom surface 315 and the firstoptically transparent wall 319 may be greater than the distance d1 ofthe integrated illumination-detection flow cell 300 of FIG. 3.

The light through each coupling optic 430 in light paths 417 may beincident on the first optically transparent wall 319 along the flowchannel 306. Upon entering the flow channel 306 through the firstoptically transparent wall 319, the light may be partly absorbed by theliquid sample flowing inside the flow channel 306. The unabsorbed lightpasses out of the flow channel 306 through the second opticallytransparent wall 318, opposite the first optically transparent wall 519,and into the integrating detection chamber 302, where it may be detectedby UV detector 305 and/or VIS detector 304, as described above withreference to FIG. 3.

Thus, the embodiments of the integrated illumination-detection flow cellsystems illustrated in FIGS. 3 and 4 both have the light source and thedetection chamber integrated with the flow cell, such that the lightpath from the light source through the flow channel to the detectionchamber passes through no optical elements (FIG. 3) or through very fewoptical elements (that is, one coupling optic 430 illustrated in FIG.4). In contrast, a non-integrated light source, flow cell, and detectorsystem, such as a traditional diode array detector system or a variablewavelength detector system, may include multiple optical elementsbetween the light source and the detector (e.g., one or more mirrors,lenses, filters, slits, and beam splitters), leading to a longer opticalpath (and thus form factor of the liquid chromatography system) andnoise generation. By integrating the light source, the flow cell, andthe detector into a single unit, a smaller form factor of the detectorunit, and thus the liquid chromatography system, is achieved.Furthermore, a signal-to-noise ratio at the UV and VIS detectors may beincreased due to higher signal and spectral purity due to the shortoptical pathlength and fewer optical components. Additionally,traditional diode array detector systems and variable wavelengthdetector systems use deuterium or tungsten lamps as the light source,which have to be pre-warmed for at least 15-20 minutes before use andhave a life span of approximately 2000 hours. In contrast, an LED lightsource, such as used in the integrated illumination-detection flow cellsystems of FIGS. 3 and 4, does not need pre-warming, as the output ofthe LED is within ±2% of a nominal output at switch-on, and has anincreased life span (e.g., >10,000 hours). As a result, the integratedillumination-detection flow cell system may be used without delay andmay experience less maintenance than a traditional diode array detectorsystem and variable wavelength detector system.

An example method 500 for operating a liquid chromatography detectorsystem is illustrated in FIG. 5. In one example, the method 500 may beused to operate the integrated illumination-detection flow cell 300 orthe integrated illumination-detection flow cell 400 illustrated in FIGS.3 and 4, respectively. Instructions for carrying out the method 500 maybe executed by a controller, such as the controller 350 of FIGS. 3 and4, based on instructions stored on a memory of the controller and inconjunction with signals received by the controller from photodetectorsof the detector system, such as the photodiode 310 and the UV detector305 and/or the VIS detector 304 illustrated in FIGS. 3 and 4.

The method 500 begins at 502 by activating a light source emitting lightof a desired wavelength. For example, the light source may be an LED ora laser diode, such as each light source 312 of FIGS. 3 and 4. Thedesired wavelength may be chosen based on an analyte of interest. Forexample, 254 nm may be chosen for detecting aromatic compounds. Thecontroller may activate the light source by supplying voltage at apredetermined duty cycle to cause the light source to emit light of thedesired wavelength at a desired output level (e.g., light intensity oroptical power output). In one example, each light source of a pluralityof light sources may be activated and controlled independently and maynot be operated at the same time with the other light sources.

As light is emitted by the activated light source, the method 500proceeds to 504. At 504, the method 500 includes adjusting the outputlevel of the activated light source. The output level of the lightsource may be adjusted by the controller coupled to the light sourcebased on the signal received by the controller from a referencedetector, such as the photodiode adjacent to the activated light source,as indicated at 506. The output level of a given light source is linearwith respect to the signal generated in the paired, adjacent referencephotodiode. In one example, in an automatic power control (APC) mode, ananalog loop may be used to maintain a constant output level of the lightsource based on the signal from the reference photodiode sent to thecontroller. An analog signal generated by the reference photodiode isrelayed to the controller. Based on the analog signal received by thecontroller, the controller may modulate (e.g., increase or decrease) thelight emitted by the light source to maintain a constant output of lightincident on a liquid sample and/or a flow channel, such as on the firstoptically transparent wall 319 of the flow channel 306 illustrated inFIGS. 3 and 4.

Additionally, the output level of the light source may be adjusted basedon absorption of the light by the liquid sample inside the flow channeland based on a minimum and a maximum threshold of the detector(s), asindicated at 508. The minimum threshold of each detector may correspondto a lower limit of detection, referring to a voltage output below whichchanges in light intensity cannot be distinguished from noise, and themaximum threshold of each detector may correspond to a saturation point,referring to a voltage output above which increases in light intensitydo not increase (or, alternatively, do not linearly increase) thevoltage output of the detector. In another example, the minimumthreshold of each detector may be above the lower limit of detection andthe maximum threshold of each detector may be below the saturation pointsuch that the output level is maintained within a linear range of thedetectors. The detectors may be coupled to an integrating detectionchamber, such as detection chamber 302 of FIGS. 3 and 4. The outputlevel of the light source may be adjusted to avoid saturating the UVand/or the VIS detectors, such as by decreasing the output level whenthe detector reaches its maximum threshold. Conversely, the output levelof the light source may also be adjusted (e.g., increased) when thedetector reaches its minimum threshold to avoid a weak signal and a poorsignal-to-noise ratio. The controller may modulate the signal-to-noiseratio to an optimal range based on the UV or the VIS detector responselevel using a separate APC control loop. The output level of theselected light source may be adjusted such that the noise signal isminimized and the sample signal is maximized (thus generating an optimalsignal-to-noise ratio).

In one example, when an amount of light absorbed by the liquid sampleinside the flow cell is high, the light entering the detection chambermay be below the minimum threshold of the detectors. Hence, the outputlevel of the activated light source may be increased to increase thelight captured in the detection chamber. In another example, when theamount of light absorbed with the liquid sample is low, the lightentering the detection chamber may be above the maximum threshold of thedetectors, resulting in a saturated signal. Hence, the output level ofthe activated light source may be reduced to decrease the light capturedin the detection chamber.

The method 500 proceeds to 510 and includes measuring the lighttransmitted through the liquid sample into the detection chamber via thedetector(s). The light that is not absorbed by the liquid sample in theflow channel enters the detection chamber. The light inside thedetection chamber may be detected by the UV detector and/or the VISdetector coupled to the detection chamber, as described above withreference to FIGS. 3 and 4.

At 512, the method 500 includes generating an absorbance spectrum of theliquid sample in the flow channel. For example, the controller mayconvert the light transmitted through the liquid sample, as detected at510, to an absorbance value (e.g., according to A=−log₁₀T), which maythen be plotted against time (and/or fraction number) on a chromatogram.The absorbance spectrum may provide information about the constituentelemental makeup of the liquid sample passing through the flow cell. Inone example, a first absorbance spectrum may be generated for thetransmitted light detected by the UV detector, and a second absorbancespectrum may be generated for the transmitted light detected by the VISdetector. In some examples, both of the first and second absorbancespectra may be plotted on a single chromatogram. The method 500 thenends.

In this way, a plurality of light sources, such as LEDs, may beintegrated within a wall of a flow cell of a liquid chromatographysystem to provide a desired wavelength light. At least a portion of thelight emitted by each of the plurality of light sources may traveldirectly to a flow cell channel containing a sample without passingthrough multiple optical elements. At least a portion of the light thatreaches the flow cell channel may be absorbed by the sample inside theflow cell, the extent of absorption depending on the constituents (e.g.,components) of the liquid sample. The light not absorbed (e.g.,transmitted) by the liquid sample in the flow cell may exit the flowcell to directly enter (without passing through optical elements) anintegrated detection chamber, which may include one or morephotodetectors coupled thereto and an integrating chamber.

The integration of the light source and the detection chamber with theflow cell reduces the use of optical elements, thereby reducing the formfactor of the liquid chromatography system. In addition, the use of noor very few optical elements between the light source and the liquidsample and between the liquid sample and the detection chamber enhancesthe signal-to-noise ratio. Furthermore, the use of LEDs, which have along lifespan and do not require pre-warming before use, may increaseefficiency and reduce a cost of operating the liquid chromatographysystem.

An example provides for a liquid chromatography device. The deviceincludes a flow cell with transmissive windows or capillary walls of aflow chamber. The transmissive windows or capillary walls are configuredto house a liquid sample. The device further includes one or more LEDsor laser-diode (LD) sources integrated within the flow cell and one ormore photodiodes adjacent to the LEDs for regulating source emissionlevel. The flow cell is coupled to a detection chamber that includes asuitable detector (e.g., UV and/or VIS detector). The detection chambermay be partially formed by one of the transmissive windows or capillarywalls of the flow chamber. Thus, the detection chamber may receive lightthat is emitted from the LED/LD and passes through the liquid sample.The detection chamber may be an integrating sphere or other suitablestructure that is configured to equally direct light from virtually allangles to the detector. A housing/chassis supports the flow cell anddetection chamber assembly.

The one or more LEDs or LD sources may each be configured to emit lightof a specific wavelength. For example, a first LED may emit light at 254nm, a second LED may emit light at 280 nm, a third LED may emit light at395 nm, and a fourth LED may emit light at 525 nm. A power source may beprovided (e.g., either internally or externally to the flow cell) toprovide energy to activate the one or more LEDs or LD sources.

The device further includes a controller. The controller is configuredto activate one or more of the one or more LEDs or LD sources. In oneexample, the controller is configured to couple a selected LED or LD tothe power source to independently activate the selected LED or LD. Forexample, an input may be received for selecting a particular wavelength(e.g., 525 nm), and in response to receiving the input, the controllermay couple the LED or LD configured to output that wavelength (e.g., thefourth LED) to the power source, thus activating that LED or LD.

The intensity of light output by the activated LED or LD may becontrolled by an automatic power control (APC) circuit, in one example.The APC circuit may be a suitable feedback loop from the output of thephotodiode to the input of the LED/LD that is configured to regulate theLED/LD light output as a function of the light detected by the adjacentphotodiode, and may include suitable circuit elements, such as an inputsampling resistor, an operating amplifier, a capacitor, a tuningtransistor, and a transmitter optical subassembly.

In some examples, the controller may be configured to tune thesignal-to-noise ratio (SNR) at the detection chamber by using the outputfrom the detector as feedback to the LED/LD input. For example, thecontroller may obtain output from the detector, and if the output isabove an upper threshold or below a lower threshold, the outputintensity of the LED/LD may be adjusted.

By including a plurality of independently controllablediscrete-wavelength light sources that include integrated photodiodes inthe flow cell rather than relying on a single broad spectrum lightsource, expensive and/or bulky optical elements such as gratings, beamsplitters, and the like may be dispensed with. As such, the LED/LDsources may be close-coupled to the transmissive window or capillarywall of the flow cell without any intervening optical elements. In thisway, the device may have a relatively small form factor. However, insome examples, coupling optics (e.g., a Fresnel lens, a prism) may beprovided between the LEDs or LDs and the transmissive window of the flowcell to increase an amount of light from the LEDs/LDs that is directedto the liquid sample in the flow cell and to the detector chamber.

While FIGS. 3 and 4 show examples of integrating light sources anddetectors with a flow cell, other detector system configurations arealso possible. In particular, detector systems that enable multiplewavelength illumination may provide added sample detection flexibility.FIG. 6 shows an example detector system 600 that may be included in aliquid chromatography system, such as HPLC system 100 of FIG. 1. Thedetector system 600 includes a light source 602 coupled to (e.g.,mounted or bonded) a substrate 601, a first collimating lens (or optic)604, and a second collimating lens (or optic) 605. The substrate 601 mayinclude a suitable substrate, such as a chip on submount, TO can,C-mount, or butterfly mount. The first collimating lens 604 and thesecond collimating lens 605 are configured to direct light emitted bythe light source 602 through a flow path 610 of a flow cell 611 to asignal detector 608. For example, if the detector system 600 is includedin an HPLC system, liquid sample may flow through the flow cell 611after eluting from an HPLC column (such as column 150 of FIG. 1) andbefore reaching a fraction collector (e.g., fraction collector 170 ofFIG. 1). In the sample interrogation region of the flow path 610, theliquid sample may be exposed to the light emitted by the light source602. As used herein, “flow path” refers to a region of the flow cell(e.g., defined by a portion of a capillary) configured to flow theliquid sample and also configured to receive and pass light. As shown,the signal detector 608 is positioned parallel to the light source 602on the opposite side of the flow cell 611. Furthermore, a referencedetector (e.g., photodiode) 606 is positioned perpendicular to the lightsource 602 on the same side of the flow cell 611.

The light source 602 may be a narrow bandwidth light source, such as alight-emitting diode (LED), organic LED (OLED), or a laser diode (LD).In one example, the light source 602 may be a single emitter that emitslight of a single wavelength (or a single wavelength range, such as 620to 640 nm for an LED that emits red light). In another example, thelight emitted by the light source 602 may be of variable wavelength,such as a tunable laser diode. In still another example, the lightsource 602 may comprise a plurality of emitters, each emitter of theplurality of emitters emitting light of a single wavelength, such as anarray of LEDs (e.g., “multi-color LEDs” or “RGB-LEDs”), or of variablewavelength, such as an array of laser diodes, as further described withrespect to FIG. 12A-B. Further, the plurality of emitters may bepackaged together or separately.

The light emitted by the light source 602 passes through the firstcollimating lens 604 and the second collimating lens 605 to enter theflow path 610 of the flow cell 611 along a light path 603. In oneexample, the light path 603 may be parallel to a direction of flow asample inside the flow path 610 for a longitudinal flow cell. In anotherexample, the light path 603 may be perpendicular to the direction offlow of the sample for a transverse flow cell. The longitudinal flowcell configuration allows for a longer pathlength (without increasingthe flow cell volume or introducing cross-sectional area changes, whichwould result in peak broadening) than the transverse flow cellconfiguration, which enables more interactions to occur between theliquid sample within the flow path 610 and the light in the light path603, increasing sample absorbance. At least a part of the light enteringthe flow cell 611 is absorbed by the sample inside the flow path 610.The unabsorbed light exits the flow cell and is detected by the signaldetector 608, with an amount of light detected by the signal detector608 varying based on both an intensity of the light emitted by lightsource 602 and absorbance characteristics of the sample inside the flowpath 610. The amount of light detected by the signal detector 608 may beused to generate an absorbance spectrum of the sample, which may be usedto determine constituents of the sample.

The reference detector 606 may control an output of the light source602. In one example, the position of the reference detector 606 may beadjacent to the light source 602. In another example, the referencephotodiode may be adjacent to the collimating lenses 604 and 605. Arelative positioning of the reference detector 606 will be describedbelow with respect to FIGS. 7-9. The output of the light source 602 maybe regulated by the reference detector based on various factors,including light absorbed by the sample inside the flow cell, feedbackfrom the signal detector 608, etc., as described above with reference toFIG. 5. The output of the light source 602 may be controlled via variouscontrol loop strategies, for example, analog circuitry, digital controlalgorithms such as proportional integrative derivative (PID), etc.

FIGS. 7-9 schematically illustrate a first configuration 700, a secondconfiguration 800, and third configuration 900, respectively, of therelative positioning of optical elements within a detector system. Thefirst configuration 700, the second configuration 800, and the thirdconfiguration 900 share common features with each other and withdetector system 600 of FIG. 6, which are numbered similarly and may notbe reintroduced. For example, the first configuration 700, the secondconfiguration 800, or the third configuration 900 may be included in thedetector system 600 of FIG. 6. FIGS. 7-9 are described collectively.

Light is emitted from the light source 602 positioned on the substrate601, passing through the first collimating lens 604 and the secondcollimating lens 605 as it travels along the light path 603. In otherexamples, additional optical elements may be present along the lightpath 603, including additional lenses (e.g., ball lens, collimatinglens, Fresnel lens), collimators, light guides, and/or other optics. Thelight source 602, the first collimating lens 604, the second collimatinglens 605, the flow path 610, and the signal detector 608 are allpositioned along a common axis traversed by the light path 603. Thelight exiting the second collimating lens 605 enters the flow path 610of the flow cell 611 via a first lens or window 742. As light in thelight path 603 passes through the liquid sample within the flow path610, at least a portion of the light is absorbed by the liquid sample.Transmitted (e.g., unabsorbed) light exits the flow cell 611 through asecond lens or window 744 and is detected by the signal detector 608,which may be a variable-wavelength detector or a diode array, forexample. The signal detector 608 may output a signal (e.g., in volts oramps) that is relative to an optical power or intensity (1) of lighttransmitted through the flow path 610 (and the liquid sample therein)along the light path 603. For example, as the intensity of lighttransmitted through the flow path 610 increases, the voltage output ofthe signal detector 608 increases. The signal output by the signaldetector 608 may be received by a controller, which may store data fromthe signal detector 608 and perform various data processing actions, asdescribed further herein (e.g., such as with respect to FIG. 5).

In some conditions, the light emitted by the light source 602 mayfluctuate (e.g., in intensity and/or wavelength). For example,variations in the current supplied to the light source and/or variationsin the temperature of the light source may result in changes to theintensity and/or wavelength output by the light source. Suchfluctuations in the light source may result in erroneous sampleabsorbance measurements if not accounted for. Thus, detector systemsgenerally include a separate reference detector (e.g., photodiode) thatmeasures the light output from the light source that does not passthrough the sample. In some examples, a beam splitter may redirect aportion of the light output by the light source to the referencedetector. However, the beam splitter may add cost and complexity to thedetector unit.

Thus, as shown in the example configurations of FIGS. 7-9, the referencedetector 606 may be positioned to detect light in the detector systemthat has reflected or backscattered off of coupling optics or otherstructures in the detector system. In one example shown in FIG. 7, aportion of light may be reflected or backscattered by the secondcollimating lens 605 and the first collimating lens 604. The referencedetector 606 is positioned off-axis from the common axis of the lightsource 602, the first collimating lens 604, the second collimating lens605, the flow path 611, and the signal detector 608 and receives theportion of light that is backscattered or reflected. Thus, the portionof the light provides a reference signal. The reference detector 606 mayoperate similarly to the signal detector 608, outputting a voltagerelative to an intensity of light detected. The control system maycorrelate fluctuations in light intensity measured by the referencedetector 606 with fluctuations in light intensity measured by the signaldetector 608 to generate a reference correction, for example.

In another example illustrated in FIG. 8, the reference detector 606 maybe located between the light source 602 and the first collimating lens603, off-axis from the common axis of the light source 602, the firstcollimating lens 604, the second collimating lens 605, the flow path611, and the signal detector 608. As shown in FIG. 8, the referencedetector 606 is positioned to receive a portion of the light emitted bythe light source 602 that is reflected or backscattered by the firstcollimating lens 604. In a further example illustrated in FIG. 9, thereference detector 606 may be present adjacent to the light source 602,coupled to the substrate 601, allowing for more efficient packaging andminiaturization. Similar to the first configuration 700 of FIG. 7 andthe second configuration 800 of FIG. 8, the reference detector 606 ispositioned off-axis from the common axis of the light source 602, thefirst collimating lens 604, the second collimating lens 605, the flowpath 611, and the signal detector 608 and is configured to receive lightreflected or backscattered by the first collimating lens 604.

As described above with reference to FIGS. 5 and 6, the referencedetector 606 and/or the signal detector 608 may control an output of thelight source 602. In one example, the output of the light source 602 maybe regulated to maintain a constant response level at the signaldetector 608. As absorption of light passing through the sample insidethe flow cell increases (for example, due to molecularcomposition/concentration of the sample), the output of the light sourcealso increases to maintain constant signal at the signal detector 608.In one example, a continuous mode of output change may occur, where theoutput of the light source changes smoothly. In another example, a rangeselector mode of maintaining the output may be used, where light outputis changed from a first range/mode to a second range/mode. This allowsthe signal detector 608 to operate in a more linear range ofresponsivity curve and allows the system to obtain a wider dynamicrange, especially when analyzing high concentration samples.

In the embodiments shown in FIGS. 6-9, light passes through the flowcell longitudinally to maximize the pathlength and the associated sampleabsorbance, as per the Beer-Lambert relationship. In an alternativeexample, the light passing through the flow cell may utilize a totalinternal reflectance off walls of the flow cell to increase an effectivepathlength of photons in the light interacting with molecules of thesample inside the flow cell. In flow cells formed by materials such asmachined blocks of metal, polymers, ceramics, quartz, glass, etc., or inlab-on-chip applications where the flow cell is formed by transparentplates bonded together, the reflectance off of walls of the flow cellmay increase the effective pathlength of the light interacting with thesample inside the flow cell.

FIG. 10 discloses an embodiment of a dichroic beam combining system1000, which may be included in a liquid chromatography system (e.g.,HPLC system 100 of FIG. 1). The dichroic beam combining system 1000includes a flow cell 1002 with light sources 1010 a, 1010 b, 1010 c, and1010 d, where each of the light sources may emit a light of differentwavelength. In another example, the light sources may each emit light ofthe same wavelength. In some examples, more than four or less than fourlight sources may be present. The dichroic beam combining system 1000uses dichroic beam combining via right angle prisms, mirrors, orrhomboid plates, which each allow a higher wavelength light to passthrough and reflect a lower wavelength light. For example, the lightsources 1010 a, 1010 b, 1010 c, and 1010 d may be included in a lineararray of LED or LD light sources arranged in decreasing wavelength ordersuch that the light source 1010 a as the longest wavelength (e.g., redlight) and the light source 1010 d has the shortest wavelength (e.g., UVlight).

Light emitted from each of the light sources passes through acorresponding first collimating lens 1008 a, 1008 b, 1008 c, and 1008 dat each light source position and enters a corresponding secondcollimating lens 1006 a, 1006 b, 1006 c, and 1006 d along a light path1011. The light emitted by each light source is represented by adifferent line type (e.g., large dash for the light source 1010 a,dotted for the light source 1010 b, short dash for the light source 1010c, and solid for the light source 1010 d). The number of first andsecond collimating lenses may each be equal to the number of lightsources. The first and the second collimating lenses reduce the angularspread of light emitted from the corresponding light source.

The light exiting each of the second collimating lenses enters adichroic combining block 1004. The dichroic combining block 1004includes a plurality of plates 1005. In one example, the plates 1005 maybe optically transparent with sufficient surface figure (e.g., less than1/20^(th) of the wavelength of light emitted by the corresponding lightsource). Each plate 1005 may be physically and optically in contact withadjacent plates and may be made of material with high refractive index,such as sapphire, fused quartz, etc. In one example, the plates 1005 maybe held together by Van der Waals forces and without adhesives. Inanother example, an adhesive or other interface material with suitableoptical transmission properties at the wavelengths of interest may beused (for example, adhesives such as NOA88, fluoropolymers such asCYTOP). In still other examples, air spaces may be present between theplates. The plates 1005 may be arranged in a parallel configurationrelative to each other (e.g., with less than 1 arcsecond angulardifference).

The light exiting each of the second collimating lenses may enter thedichroic combining block 1004 through an entrance surface 1020 of eachof the plates 1005. The light from the light channel may collectivelyexit the dichroic combining block 1004 though an exit surface 1022 alonga light path 1012 and enter a flow path 1003 of a flow cell 1002. Inthis way, the light emitted separately by each light source is combinedinto a single beam. Additional focusing optics may be placed between theexit surface 1022 and the flow cell 1002. Furthermore, an additionalsurface may be provided to fold the light in the dichroic combiningblock 1004 based on a relative arrangement of the flow cell 1002, suchas due to packaging constraints. As shown in FIG. 10, a signal detector1024 is positioned on an opposite side of the flow cell 1002 from thedichroic combining block 1004 and on a common axis with the flow path1003 and the light path 1012.

The plates 1005 may include dichroic coating(s) to redirect the lightbeams entering the light channel to be collinear. Additional dichroiccoatings may be added to the entrance surface and the exit surface ofthe light channel to decrease reflective loss of light.

Each of the optical elements illustrated in FIG. 10 may be positionedand secured on a structural chassis, for example, by adhesives and/or bymechanical means. The example of FIG. 10 shows four plates 1005 for fourlights sources 1010 a, 1010 b, 1010 c, and 1010 d, but in otherexamples, two or more plates 1005 may be included to combine lightemitted by three light sources.

In another embodiment, multiple dichroic combining blocks may bearranged in parallel. For example, a first set of four light sources mayfeed a first dichroic combining block, and a second set of four lightsources may feed a second dichroic combining block. The light combinedby the first dichroic combining block and the light combined by thesecond dichroic combining block may be directed to a third dichroiccombining block to achieve a combined eight wavelengths of light, whichmay then be directed to a flow cell. Such a configuration allows forselection of simplified optical filters to combine sources with peakwavelengths that are close to each other (e.g., within a smallwavelength difference).

Dichroic combining enables efficient and collinear combining of multiplewavelengths of light, resulting in a smaller form factor, which is idealfor portable systems. The dichroic combining may provide two to eightwavelength of light without changing chassis size. The configuration fordichroic combining described above provides a fixed alignment, which isstable against vibration and shock loads.

FIG. 11 shows a fiber-coupled beam combining system 1100, which may beincluded in a liquid chromatography system (e.g., HPLC system 100 ofFIG. 1). The fiber-coupled beam combining system 1100 uses opticalfibers to achieve coaxial beam combining. Each of a plurality of lightsources 1112 is positioned on a corresponding substrate 1113. In oneexample, each light source 1112 may emit a light of a differentwavelength. In another example, each light source may emit a light of asame wavelength. In one example, each of the plurality of light sources1112 may be an LED or an LD light source. As an example, when each ofthe plurality of light sources 1112 emits light of the same wavelength,the plurality of light sources 1112 may be lower wavelength LEDs (e.g.,255 nm or 230 nm), which emit lower power light than higher wavelengthLEDs.

In one example, each of the light sources 1112 may be coupled to acorresponding, separate substrate 1113 while in another example, theplurality of light sources 1112 may be coupled to a common substrate. Inanother example, two or more light sources may be coupled to eachsubstrate 1113. In further embodiments, the substrate and the lightsources may be present in a different ratio. The substrates 1113 may bemade of low-cost materials, for example, metal core circuit boards,allowing for reduced costs and greater flexibility in configurations ofthe substrates. Each of the light source and substrates may be spaced asneeded for optimal thermal dissipation and/or to meet packagingconstraints.

Each of the light sources 1112 may be coupled to a corresponding opticalfiber 1108 at a joining node 1110. The coupling at the joining node 1110may be a mechanical coupling, through an adhesive, or through afused-fiber combiner. Each of the optical fibers 1108 may join at acommon node 1106. A coaxial beam 1104 emitted from the common node 1106is delivered to a flow path 1103 of a flow cell 1102. The optical fibers1108 may be spaced for a compact optical path to the flow cell, forexample. As shown in FIG. 11, a signal detector 1124 is positioned on anopposite side of the flow cell 1102 from the common node 1106 and on acommon axis with the flow path 1103 and the coaxial beam 1104.

FIG. 12A shows a waveguide beam combining system 1200 with a waveguide1206 for combining light beams and directing the combined light beam toa flow cell 1202. The waveguide beam combining system 1200 includes asubstrate 1209 with an arrangement of light sources 1208. In oneexample, the arrangement of light sources 1208 coupled to the substrate1209 may include four light sources (e.g., LEDs or LDs) placed in a twoby two array, as illustrated in FIG. 12B. Each of the light sources inthe arrangement of light sources 1208 may emit light of a differentwavelength. In other examples, the number and the arrangement of lightsources on the substrate 1209 may vary.

The arrangement of light sources 1208 is positioned at an entrance 1205of the waveguide 1206, as illustrated in FIG. 12A. Light emitted fromthe arrangement of light sources 1208 travels along the waveguide 1206and exits the waveguide at an exit 1207 to enter a collimating optic1204 along a light path 1211. In one example, more than one collimatingoptic may be present between the waveguide 1206 and the flow cell 1202.The light exiting the collimating optics 1204 enters a flow path 1203 ofthe flow cell 1202 and travels along a longitudinal length of the flowpath 1203 containing a sample. The light exiting the flow path 1203 ofthe flow cell 1202 is detected by a signal detector 1210.

The waveguide 1206 may be hollow and cylindrical, triangular, square,hexagonal, or any other shape to allow the light beam to enter throughthe entrance and travel through the waveguide to the exit. In oneexample, the waveguide 1206 may include reflective inner walls and maybe filled with a suitable gas or may have a vacuum to enable optimallight mixing. In one example, the inner walls of waveguide 1206 maycomprise aluminum surface mirrors.

The waveguide beam combining system 1200 delivers a very homogenouslight to the flow cell 1202 after mixing inside the waveguide 1206. Thelight entering the waveguide includes light of various wavelengthsemitted from each of the light sources in the arrangement of lightsources 1208. As the light travels along the waveguide 1206, the lightof the various wavelengths mixes such that the light exiting thewaveguide has a uniform mixture of light of the various wavelengths. Theuse of commercially available components and simple alignments of thecomponents of the waveguide beam combining system 1100 allows for lowcost manufacturing.

FIG. 13 shows an integrating chamber beam combining system 1300 thatincludes an integrating chamber 1306 for combining a multiplewavelengths of light into a single light beam before the light beam ispassed through a flow path 1303 of a flow cell 1302.

Each light source of an arrangement of light sources 1308 coupled to asubstrate 1309 emits light, which enters the integrating chamber 1306.In one example, the arrangement of light sources 1308 coupled to thesubstrate 1309 may include four light sources (e.g., LEDs or LDs) placedin a two by two array, such as the arrangement of light sources 1208 asillustrated in FIG. 12B. Each of the light sources in the arrangement oflight sources 1308 may emit light of a different wavelength. In otherexamples, the number and the arrangement of light sources on thesubstrate 1309 may vary. In one example, the light sources 1308 may bepositioned along a perimeter of the integrating chamber 1306.

Inner surfaces of the integrating chamber 1306 may be made of areflective material with high reflectivity over a desired wavelengthrange. In one example, when a large wavelength range is desired, adiffusely reflective material that can reflect light ranging from 200 nminto to the visible light range may be used, such aspolytetrafluoroethylene (PTFE). In another example, a specularlyreflective surface may be used, such as uncoated aluminum may be usedinside the integrating chamber 1306. The integrating chamber 1306 may bea hemisphere, similar to half of an integrating sphere. Other geometriesfor the integrating chamber 1306 may also be suitable.

The light that exits the integrating chamber is of homogeneouswavelength due to mixing within the integrating chamber 1306. The lighttravels along an optical fiber 1304 and is directed along light path1312 to enter the flow path 1303 of the flow cell 1302. In one example,more than one optical fiber may carry the light from the integratingchamber towards the flow cell. The light exiting the flow cell 1302 isdetected by a signal detector 1310. In one example, one or morecollimating optics may be present between the integrating chamber 1306and the flow cell 1302 to direct light to the flow path 1303 of the flowcell 1302.

A polygonal prism beam combining system 1400 with a prism beam combiner1404 that combines light emitted from a plurality of light sources 1420is illustrated in FIG. 14. The prism beam combiner 1404 may be amonolithic transparent polygon with a flat input surface 1410 for eachcorresponding light source 1420 and one flat output surface 1411 fordirecting a mixed light beam 1406 to a flow path 1403 of a flow cell1402. In one example, the prism beam combiner may be made of reflectiveoptical plates with dichroic coating.

In one example, the prism beam combiner 1404 may be a pentagon with fiveflat surfaces, of which four flat input surfaces may receive lightemitted from the corresponding light source while a fifth flat outputsurface of the prism beam combiner faces the flow cell 1402 to directthe mixed light beam 1406 from the prism beam combiner 1404 to the flowpath 1403. In another example, the prism beam combiner 1404 may have sixflat surfaces, where five of the six flat surfaces receive light fromthe corresponding light sources and the mixed light beam 1406 isdirected through a sixth flat surface to the flow path 1403. As such,any number of sides may be chosen for the polygon such that a totalnumber of sides is one greater than a number of light sources.

Each light source 1420 is coupled to a substrate 1421. The light emittedby each of the light sources 1420 travels along a corresponding lightpath 1422 through a corresponding first collimating lens 1414 and acorresponding second collimating lens 1416 and is incident on acorresponding flat input surface 1410 of the prism beam combiner 1404.The light emitted by each of the light sources 1420 is illustrated bydifferent line types (e.g., solid, short dash, long dash, and dotted).The light from each of the light sources 1420 mixes inside the prismbeam combiner 1404 and exits the flat output surface 1411 as the mixedlight beam 1406 to enter the flow path 1403, where it may be at leastpartially absorbed by a liquid sample inside of the flow path beforebeing transmitted to a signal detector 1424. The prism beam combiner1404 along with light sources 1420 and the flow cell 1402 may all bepositioned along a mechanical chassis, which may enable fixed alignmentthat is stable against vibration and shock. The prism beam combinerenables combining of multiple wavelengths of light without increasing asize of the prism.

FIG. 15 shows a compound beam combining system 1500, including aplurality of optical fibers 1508 coupled to an integrating rod 1506. Theintegrating rod 1506 is coupled to a total internal reflection (TIR)optic 1504. A plurality of light sources 1520, each coupled to arespective substrate 1521, emit light. Each of the light sources 1520may be LEDs emitting light of a same wavelength in one example. Inanother example, each of the LEDs may emit light of a differentwavelength. Each of the light sources 1520 coupled to the correspondingsubstrate 1521 may be spatially separated from each other to lower heatflux density and for efficient thermal management.

The light emitted from each of the light sources 1520 is directedthrough a corresponding optical fiber 1508 coupled to the light sourceat a joint 1524. In one example, an adhesive may couple (e.g., bond) thecorresponding optical fiber 1508 to the each of the light sources 1520.In another example, the coupling may be mechanical (e.g., a butt joint).Each of the optical fibers 1508 may be a large core, large numericalaperture multimode optical fiber, for example.

The light travelling through each of the optical fibers 1508 enters theintegrating rod 1506, which results in mixing of the light inside theintegrating rod 1506. In one example, the integrating rod 1506 may bemade of fused silica. In another example, a reflective-walledintegrating rod or integrating chamber may be used to mix the beam oflight received through the optical fibers 1508. In one example, walls ofthe integrating rod may be made of UV-transmittance aluminum coatedsurface mirrors.

The TIR optic 1504 images light exiting the integrating rod 1506. TheTIR optic includes a first elliptical focal point 1504 a that ispositioned at a center of an exit plane of the integrating rod 1506 onan outer surface of the TIR optic 1504. The first elliptical focal point1504 a images (e.g., directs) the high angle/high numerical aperturerays to a second elliptical focal point 1504 b positioned at an opticalinlet of the flow cell 1502, effectively reducing the angle of the highnumerical aperture rays. A third optical surface of the TIR optic,located internally, may be substantially planar and images low-anglerays exiting the integrating rod 1506. The light exiting the TIR opticenters a flow path 1503 of a flow cell 1502 along a light path 1522along a longitudinal length of the flow cell.

In one example, the TIR optic may be a diamond-turned crystalline. Inanother example, the TIR optic may be a molded material such as calciumfluoride or magnesium fluoride, fused quartz, a high-UV transmissionsilicone, or a fluoropolymer.

In another example, a reflecting optic and a separate lens may be usedin place of the TIR optic 1504. In another example, a fused fibercombiner may be used in place of the optical fibers 1508 and theintegrating rod 1506.

FIG. 16 illustrates a multiplexing system 1600 for directing light totravel through a flow cell (not shown). The multiplexing system 1600includes a reflective surface 1604 that rotates along an axis 1601. Inone example, the reflective surface may be a mirror. In another example,the reflective surface 1604 may be a reflective prism. In a furtherexample, the reflective surface 1604 may be of a two-axis gimballedmirror.

The reflective surface 1604 may be positioned on a stage 1610 that maybe rotated along the axis 1601. The stage 1610 may be coupled through anaxle 1608 to a motor 1612. The motor 1612 may be operated to rotate thestage and hence, the reflective surface 1604 at a desired rotation speedin a clockwise and/or a counterclockwise direction. In one example, thereflective surface 1604 may be rotated at a constant speed. In anotherexample, the reflective surface 1604 may be indexed from one position toa next position. The motor 1612 may be a galvanometer, a limited anglemotor, or other suitable motor. Noise generated due to rotation by themotor may be reduced via a lock-in amplifier (not shown) coupled to themultiplexing system 1600. In another example, a micro machined MicroOptical Electrical Mechanical Systems (MOEMS) mirror may be used inplace of the reflective surface 1604 and the motor 1612.

A plurality of light sources 1620 are placed radially around a perimeterof the stage 1610. The light sources 1620 may be LED or LD lightsources, where each light source may emit light of a differentwavelength. A number light sources in the plurality of light sources1620 may vary. For example, eight light sources 1620 are illustrated inFIG. 16. In other examples, the number of light sources may be less thanor more than eight. In one example, each of the light source may bespaced and angled similarly relative to the reflective surface 1604. Inanother example, the light sources 1620 may be placed at staggereddistances and differing angles relative to the reflective surface 1604to meet packaging constraints. Each of the light sources may beindividually selected to emit light of a desired wavelength.

Based on the rotation of the stage with the reflective surface 1604 andalignment of a selected light source 1620 with the reflective surface1604, light emitted by the selected light source 1620 may be incident onthe reflective surface 1604. Light reflected from the reflective surfacemay then be directed to a flow cell with a sample (not shown). One ormore collimating optics may be present between each of the light sources1620 and the reflective surface 1604 and between the reflective surface1604 and the flow cell (not shown). In an alternative example, thereflective surface 1604 may be stationary while the light sources 1620may be rotatable such that light emitted from the selected light source1620 is incident on the reflective surface 1604. The multiplexing system1600 may enable temporal switching of the selected light source 1620depending on characteristics of the sample inside the flow cell.

In this way, various configurations of optical elements described abovewith reference to FIGS. 6-16 may be used in a liquid chromatography flowcell for increasing packaging efficiency, for dissipating excess heat,for homogenous mixing of light from multiple light sources, and forstable and cost-effective alignment of optical elements for accuratesample analysis.

As one example, a detector system comprises: a flow cell including anoptically transparent first wall and an optically transparent secondwall, the optically transparent second wall positioned opposite theoptically transparent first wall; a plurality of light sourcesintegrated within the flow cell, the plurality of light sourcesconfigured to emit light to travel through the optically transparentfirst wall into the flow cell; and a detection chamber integrated withthe flow cell and configured to capture light passing out from the flowcell through the optically transparent second wall into the detectionchamber. In the preceding example, additionally or optionally, aninterior of the detection chamber includes a diffuse, reflective coatingthat uniformly scatters the captured light. In any or all of thepreceding examples, additionally or optionally, each of the plurality oflight sources is adjacent to a reference photodiode integrated withinthe flow cell, the reference photodiode configured to detect lightemitted by the adjacent light source. In any or all of the precedingexamples, the detector system additionally or optionally furthercomprises a photodetector coupled to the detection chamber. In any orall of the preceding examples, additionally or optionally, thephotodetector is configured to detect ultraviolet and/or visible light.In any or all of the preceding examples, the detector systemadditionally or optionally further comprises a coupling optic betweeneach of the plurality of light sources and the first transparent wall ofthe flow cell. In any or all of the preceding examples, additionally oroptionally, no coupling optics are included in the detector system. Inany or all of the preceding examples, additionally or optionally, thedetector system is included in a high-performance liquid chromatographysystem. In any or all of the preceding examples, the detector systemadditionally or optionally further comprises a controller storingexecutable instructions in non-transitory memory that, when executed,cause the controller to: activate a light source of the plurality oflight sources to emit light of a desired wavelength; and control anoutput level of the activated light source based on input received fromthe reference photodiode adjacent to the light source.

As another example, a method comprises: activating one light emittingdiode (LED) from a plurality of LEDs integrated within a flow cell;adjusting an output of the one LED based on a signal from a referencephotodiode adjacent to the one LED, the output of the one LED passingthrough a first optically transparent wall of the flow cell into asample chamber of the flow cell; and detecting an amount of lighttransmitted through the sample chamber through a second opticallytransparent wall of the flow cell, opposite the first opticallytransparent wall, via one or more photodetectors coupled to a detectionchamber integrated with the flow cell. In the preceding example,additionally or optionally, each of the plurality of LEDs emits light ofa different wavelength. In any or all of the preceding examples,additionally or optionally, the detection chamber is an integratingchamber, and a bottom surface of the detection chamber is at leastpartially comprised of the second optically transparent wall. In any orall of the preceding examples, the method additionally or optionallyfurther comprises determining an absorbance of a sample within thesample chamber based on the detected amount of light transmitted throughthe sample chamber. In any or all of the preceding examples,additionally or optionally, adjusting the output of the one LED based onthe signal from the reference photodiode adjacent to the one LEDincludes modulating the output of the one LED so that an amount of lightmeasured by the reference photodiode remains constant. In any or all ofthe preceding examples, the method additionally or optionally furthercomprises adjusting the output level of the one LED based on a signalmeasured by the one or more photodetectors based on a signal-to-noiseratio.

As another example, a multiple wavelength illumination system comprises:a flow cell configured to receive light emitted from a plurality oflight sources; at least one light combining device to mix the lightemitted by the plurality of light sources before the light is receivedby the flow cell; at least one collimating optic to direct light fromthe at least one light combining device to the flow cell; and a detectorto detect light exiting the flow cell. In the preceding example,additionally or optionally, wherein the at least one light combiningdevice includes a fiber optic cable. In any or all of the precedingexamples, additionally or optionally, the at least one light combiningdevice includes a waveguide. In any or all of the preceding examples,additionally or optionally, the at least one light combining device hasa dichroic coating. In any or all of the preceding examples,additionally or optionally, the at least one light combining deviceincludes a polygonal prism.

As used herein, an element or step recited in the singular and proceededwith the word “a” or “an” should be understood as not excluding pluralof said elements or steps, unless such exclusion is explicitly stated.Furthermore, references to “one embodiment” of the present invention arenot intended to be interpreted as excluding the existence of additionalembodiments that also incorporate the recited features. Moreover, unlessexplicitly stated to the contrary, embodiments “comprising,”“including,” or “having” an element or a plurality of elements having aparticular property may include additional such elements not having thatproperty. The terms “including” and “in which” are used as theplain-language equivalents of the respective terms “comprising” and“wherein.” Moreover, the terms “first,” “second,” and “third,” etc. areused merely as labels, and are not intended to impose numericalrequirements or a particular positional order on their objects.

FIGS. 1-4 and FIGS. 6-16 show example configurations with relativepositioning of the various components. If shown directly contacting eachother, or directly coupled, then such elements may be referred to asdirectly contacting or directly coupled, respectively, at least in oneexample. Similarly, elements shown contiguous or adjacent to one anothermay be contiguous or adjacent to each other, respectively, at least inone example. As an example, components laying in face-sharing contactwith each other may be referred to as in face-sharing contact. Asanother example, elements positioned apart from each other with only aspace therebetween and no other components may be referred to as such,in at least one example. As yet another example, elements shownabove/below one another, at opposite sides to one another, or to theleft/right of one another may be referred to as such, relative to oneanother. Further, as shown in the figures, a topmost element or point ofelement may be referred to as a “top” of the component and a bottommostelement or point of the element may be referred to as a “bottom” of thecomponent, in at least one example. As used herein, top/bottom,upper/lower, above/below, may be relative to a vertical axis of thefigures and used to describe positioning of elements of the figuresrelative to one another. As such, elements shown above other elementsare positioned vertically above the other elements, in one example. Asyet another example, shapes of the elements depicted within the figuresmay be referred to as having those shapes (e.g., such as being circular,straight, planar, curved, rounded, chamfered, angled, or the like).Further, elements shown intersecting one another may be referred to asintersecting elements or intersecting one another, in at least oneexample. Further still, an element shown within another element or shownoutside of another element may be referred as such, in one example.

This written description uses examples to disclose the invention,including the best mode, and also to enable a person of ordinary skillin the relevant art to practice the invention, including making andusing any devices or systems and performing any incorporated methods.The patentable scope of the invention is defined by the claims, and mayinclude other examples that occur to those of ordinary skill in the art.Such other examples are intended to be within the scope of the claims ifthey have structural elements that do not differ from the literallanguage of the claims, or if they include equivalent structuralelements with insubstantial differences from the literal languages ofthe claims.

1. A detector system, comprising: a flow cell including an opticallytransparent first wall and an optically transparent second wall, theoptically transparent second wall positioned opposite the opticallytransparent first wall; a plurality of light sources integrated withinthe flow cell, the plurality of light sources configured to emit lightto travel through the optically transparent first wall into the flowcell; and a detection chamber integrated with the flow cell andconfigured to capture light passing out from the flow cell through theoptically transparent second wall into the detection chamber.
 2. Thedetector system of claim 1, wherein an interior of the detection chamberincludes a diffuse, reflective coating that uniformly scatters thecaptured light.
 3. The detector system of claim 1, wherein each of theplurality of light sources is adjacent to a reference photodiodeintegrated within the flow cell, the reference photodiode configured todetect light emitted by the adjacent light source.
 4. The detectorsystem of claim 1, further comprising a photodetector coupled to thedetection chamber.
 5. The detector system of claim 3, wherein thephotodetector is configured to detect ultraviolet and/or visible light.6. The detector system of claim 1, further comprising a coupling opticbetween each of the plurality of light sources and the first transparentwall of the flow cell.
 7. The detector system of claim 1, wherein nocoupling optics are included in the detector system.
 8. The detectorsystem of claim 1, wherein the detector system is included in ahigh-performance liquid chromatography system.
 9. The detector system ofclaim 3, further comprising a controller storing executable instructionsin non-transitory memory that, when executed, cause the controller to:activate a light source of the plurality of light sources to emit lightof a desired wavelength; and control an output level of the activatedlight source based on input received from the reference photodiodeadjacent to the light source.
 10. A method, comprising: activating onelight emitting diode (LED) from a plurality of LEDs integrated within aflow cell; adjusting an output of the one LED based on a signal from areference photodiode adjacent to the one LED, the output of the one LEDpassing through a first optically transparent wall of the flow cell intoa sample chamber of the flow cell; and detecting an amount of lighttransmitted through the sample chamber through a second opticallytransparent wall of the flow cell, opposite the first opticallytransparent wall, via one or more photodetectors coupled to a detectionchamber integrated with the flow cell.
 11. The method of claim 10,wherein each of the plurality of LEDs emits light of a differentwavelength.
 12. The method of claim 10, wherein the detection chamber isan integrating chamber, and a bottom surface of the detection chamber isat least partially comprised of the second optically transparent wall.13. The method of claim 10, further comprising determining an absorbanceof a sample within the sample chamber based on the detected amount oflight transmitted through the sample chamber.
 14. The method of claim10, wherein adjusting the output of the one LED based on the signal fromthe reference photodiode adjacent to the one LED includes modulating theoutput of the one LED so that an amount of light measured by thereference photodiode remains constant.
 15. The method of claim 10,further comprising adjusting the output level of the one LED based on asignal measured by the one or more photodetectors based on asignal-to-noise ratio.
 16. A multiple wavelength illumination system,comprising: a flow cell configured to receive light emitted from aplurality of light sources; at least one light combining device to mixthe light emitted by the plurality of light sources before the light isreceived by the flow cell; at least one collimating optic to directlight from the at least one light combining device to the flow cell; anda detector to detect light exiting the flow cell.
 17. The multiplewavelength illumination system of claim 16, wherein the at least onelight combining device includes a fiber optic cable.
 18. The multiplewavelength illumination system of claim 16, wherein the at least onelight combining device includes a waveguide.
 19. The multiple wavelengthillumination system of claim 16, wherein the at least one lightcombining device has a dichroic coating.
 20. The multiple wavelengthillumination system of claim 16, wherein the at least one lightcombining device includes a polygonal prism.